Aims: In post-operative and post-trauma therapy of fluid imbalance, intravenous hyperoncotic albumin is widely used for plasma volume expansion. However, possible deleterious effects of hyperoncotic albumin on endothelial permeability are reported, but not understood. This application is to determine basic mechanisms which cause such permeability effects. We propose that hyperoncotic albumin increases transendothelial, vesicular transport of albumin and thereby increases transendothelial liquid flux. The vesicular transport may be activated by increases of endothelial Ca++(Eca) which lead to cytoskeletal reorganization. To test these hypotheses, we will use novel techniques in the single lung capillary to quantify endothelial hydraulic conductivity (Lp), endothelial vesicular transport (Jves), and Eca. In cultured endothelial cells, we will determine endothelial cytoskeletal responses through imaging and western blotting studies. Specific inhibitors of the signal transduction pathway will be used, to determine cellular mechanisms in the permeability response. Procedures: The single microvessel protocols are planned in isolated blood perfused rat lungs. Bright field and fluorescent microscopic images of subpleural capillaries will be analyzed by computer based digital image analysis. By micropuncture, the selected microvessel will be first preinfused with experimental solutions. The (i) for Lp measurements by our new split drop technique, an oil drop microinjected in the microvessel will be split with a standard solution. Transendothelial flux (Jv) of the splitting solution will be interpreted from timed measurements of the oil drop movement, at different capillary pressures (Pc). Lp will be determined as the slope of the Jv-Pc line; (ii) for Jves measurements, the capillary will be injected with fluorescent ligands, unbound fluorescence will be washed out by blood flow and endothelial fluorescence will be digitally quantified with respect to time; (iii) Eca determinations in single capillaries will be obtained by the Fura technique; (iv) for in vitro protocols, cultured endothelial cells will be prepared then exposed to albumin concentrations. Tyrosine kinase activation will be determined by western blotting with anti- phosphotyrosine antibody. The cytoskeleton will be imaged by fluorescent techniques. Significance: If preliminary data bear out, this research will prove the first time that hyperoncotic albumin increases lung capillary permeability. Such findings will engender the important understanding that the intravenous use of concentrated albumin predisposes to pulmonary edema. Important new basic advances will be achieved in the understanding of endothelial Ca++ and the endothelial cytoskeleton in the regulation of transendothelial vesicular transport. The use of the in situ capillary will advance understanding of cell biological responses in an intact system. As such, this research will lead to a fundamentally new understanding of lung endothelial barrier regulation.